Malignant primary brain tumors represent the most frequent cause of cancer death in children and young adults and account for more deaths than cancer of the kidney or melanoma. Glioblastoma (GBM), the most common of these tumors, is uniformly lethal. Moreover, current therapy is non-specific and produces a median overall survival of only <15 months. In contrast, immunotherapy promises an exquisitely precise approach, and substantial evidence suggests that T cells can eradicate large, well-established tumors in mice and humans even when tumors reside within the brain. Notably, tumor immunotherapy has been recently validated in phase III clinical trials, leading to pivotal approvals by the FDA of at least two prototypic immune-based cancer treatments within the past two years. Bispecific T cell Engagers (BiTEs) represent an emerging class of bispecific antibody that has been shown to effectively redirect T cells against tumor cells. BiTEs promise to overcome many critical barriers that have traditionally limited translation of immunotherapy to the clinic. Separating this platform from other available immunotherapeutic approaches, our preliminary data support that BiTEs are (1) highly-specific molecules that greatly reduce the risk of toxicity, (2) have the ability to penetrate the blood-brain barrier (BBB) and accumulate in intracerebral tumors, and (3) may potentially overcome multiple mechanisms of immunosuppression present in patients with GBM. To date, BiTEs have not previously targeted tumor-specific antigens, and until our studies, have not yet been tested for their ability to mediate activity against tumors of the central nervous system (CNS). Here, we have designed a BiTE against the EGFRvIII tumor-specific antigen and perform preclinical tests to determine its efficacy against EGFRvIII- expressing GBM. In Aim 1 of this proposal, we will evaluate the risk of toxicity and validate efficacy of the EGFRvIII-specific BiTE in a novel human CD3 transgenic mouse background. The unique model, along with a murine homologue of human EGFRvIII that we have created, will allow us to evaluate the actual construct we plan to take into clinic. Moreover, because the CD3 transgenic mice are heterozygotes and have a normal immune response, this model will allow us to assess the development of secondary autoimmunity and the potential for development of endogenous immune responses against tumor cells that do not express EGFRvIII. In Aim 2, we will explore the possibility that, unlike other macromolecules, BiTEs have the unique ability to penetrate an intact BBB and determine whether this effect can be modulated to improve antitumor efficacy. Finally, in Aim 3, we will conduct early clinical studies to assess biodistribution, early toxicity, and potential efficacy of the EGFRvIII-specific BiTE in patients with EGFRvIII-expressing GBM. Overall, this work has the potential to improve the clinical management of patients with GBM by generating a novel therapeutic.